The overall goals of this NCDDG application is to discover novel anticancer drugs based on inhibiting the aberrant activation of Rho GTPases in human cancer. Aberrant activation of Rho GTPases is associated with tumor invasion, metastasis and poor prognosis. Aberrant activation involves several mechanisms including Rho overexpression (RhoC), alternative splicing (Rac1) or persistent activation of guanine nucleotide exchange factor (RhoGEFs such as the Rac specific GEF Tiam1, and the RhoA/C specific GEF, LARG). Furthermore, persistent activation of Rho GTPases depends on their posttranslational modification by geranylgeranyltransferase 1 (GGTase 1) anci the transforming activity of some Rho GTPases requires the ser/thr kinase Rho kinase (ROCK). The hypothesis upon which this NCDDG is based is that inhibitors of GEFs, GGTase I and RhoK will reverse malignant transformation of cancers with aberrant Rho function. To test this hypothesis we propose 3 complementary and interdependent programs and 2 cores. Program 1 will design, based on available structural information as well as hits from Core B, novel GGTase I, GEF and RHOK inhibitors. Core B will use experimental and virtual high throughput screening (HTS) as well as molecular modeling to identify GGTase I, RhoGEF and ROCK inhibitors. Program 2 will focus on validation and characterization of inhibitors of RhoGEF, GGTase I and RHOK. Program 3 will evaluate the effects of GEF, GGTase I and RHOK inhibitors on Rho signaling and function as well as their antitumor activity, pharmacokinetics and toxicity in animal models. The 3 programs and the 2 cores will work very closely together towards achieving the specific objectives and the overall goal of the NCDDG. These concerted efforts will lead to novel anticancer drugs that will increase the spectrum of human cancers that can be treated by thwarting the aberrant activation of Rho GTPases.